Packaging for biologival mateial

ABSTRACT

A packaging for biological material comprises two substantially parallel walls connected to each other along a part of their periphery and in a central area. The packaging may be used for cryopreservation of biological material.

The present invention relates to a packaging for storing, transportand/or freezing of a biological material such as fish semen or milt.More particular, the invention regards a packaging for cryopreservationof spermatozoa, embryos or eggs from aquatic organisms.

Traditionally, semen or milt are cryopreserved in cryogenic strawconsisting of a narrow and slender tube as for example disclosed in EP 0873 726 A1, EP 0,562,947 A1, U.S. Pat. No. 5,868,178, and U.S. Pat. No.5,190,880. The main drawback with these straws is the fact that onlysmall volumes of biological material can be filled into these straws.

Usually, 0.5-1.2 ml semen are frozen in conventional medium cryogenicstraw. Trials have been performed freezing 5 ml in comparison with 1.2ml, resulting in a dramatic decrease in fertilization success(Lahnsteiner et al, 1997: Aquaculture Research 28, 471-479). Theaquaculture industry need an efficient large scale cryopreservationprotocol and thus preferably a method and means which enablescryopreservation of larger amounts of semen.

Furthermore, it is a well accepted fact that larger and less narrowstraw would not be useful since they cannot provide sufficient uniformfreezing of the content (e.g. semen), and thus causes reduced quality ofthe cryopreserved material.

For animal semen, pouches have been proposed for packaging and laterartificial insemination of animals. One example of such a pouch is shownin U.S. Pat. No. 7,150,734 and DK/EP 0605406. However, these pouches arenot suited for cryopreservation, as the volume is too large to ensureuniform freezing of the full content.

The object of the invention is to provide a packaging for biologicalmaterial which contains a significant volume while ensuring a uniformtemperature of the whole content, for example during freezing andthawing. A significant volume in this context will depend on the type ofbiological material, for example varying between species, but is of atleast 5 ml.

In the following description, the advantages of the invention isdescribed when used in connection with freezing the biological material,for example for cryopreservation, but it will be obvious for the skilledperson that the packaging according to the invention can be used forother purposes such as long and short time storage and transport.

The object of the invention is achieved by means of the features of thepatent claims.

According to the invention, there is provided a packaging for biologicalmaterial which comprises two substantially parallel walls connected toeach other along a substantially part of their periphery and in at leasta central area.

The biologic material is for example semen, embryos, eggs, etc. Theinvention is particularly suitable for cryopreservation of spermatozoacollected from aquatic species. Aquatic species are in this context anyorganism living in water and wherein reproduction is performed by thejoining of spermatozoa and egg from a male and female animal,respectively, herein organisms employing oviparous, ovoviviparous orviviparous reproduction. The present invention allows packaging of malegametes (sperm) collected from marine, anadromous and fresh water fish.However, the invention is not limited to fish species, but also includesother water-dwelling organisms such as Crustacea, inter alia crabs,lobsters, crayfish, shrimps, shellfish and barnacles, and Mollusca,inter alia gastropods such as abalone, cephalopods such as octopus, andbivalves such as scallops, clams and oysters.

Non-limiting examples of oviparous fish species are inter aliaSalmonidae, such as Atlantic salmon (Salino salar L.) and rainbow trout(Onchorhyncus mykiss), Gadidae, such as Atlantic cod (Gadus morhua) andhaddock (Melanogrammus aeglefinus) and Cichlidae, such as Nile tilapia(Oreochromis niloticus niloticus). Non-limiting examples ofovoviviparous fish include inter alia Poecilidae, such as guppy(Poecilia reticulata) and Squatinidae, such as angel sharks (Squatinaspp.). Non-limiting examples of viviparous fishes includes coelacanths(Latimeria chalumnae), Goodeidae (splitfins), Embiotocidae(surf-perches) and Carcharhinidae (requiem sharks).

The parallel arrangement of the walls provides an evenly distribution ofthe biological material to be frozen. The distance separating the wallsdecides the thickness of the layer of biologic material, thus a smalldistance provides a thin layer of biological material and thus a uniformfreezing of the content. The walls may have any shape and may bestraight or not, i.e. wavy, as long as they are parallel and provide anevenly distribution of a thin layer of e.g. semen/milt.

In one embodiment, the walls are separated by a distance between theinner surface of the two walls which is about 2 mm, for example about1,1 mm, about 1.0 mm, or about 0,95 mm.

The distance separating the walls depend on which cooling rate that iscontemplated, the material of the device, to which degree the device isfilled etc. The distance may also be about 1 or 2 mm. Furthermore, byusing a flexible material which enables a certain expansion of thedevice upon freezing of the contained semen, the distance may vary fromabout 0.95-2 mm

The biological material, such as sperm cells is thus evenly and sparselydistributed between the walls of the device. The device according to thepresent invention thus provides an equal temperature curve for all thesperm cells contained in the present device throughout a full freezingand thawing period. Such evenly and sparsely distribution andcorresponding equal temperature curves may be obtained also by use ofconventional cryogenic straw consisting of a narrow and slender tube.However, equal temperature curves for all cells during the entirefreezing and thawing procedure will not be possible to obtain withconventional straw when the volume of each straw is increased beyond 0.5ml.

The packaging may have one or more openings. The openings may bearranged for filling the packaging by means of a filling device,applying suction, draining air, etc.

An opening for filling the packaging may be provided any suitable placein the packaging. In one embodiment the opening is placed in theperiphery of the packaging, for example in one side. The opening may bein a closed state prior to filling the packaging, thus preventingcontamination of the inner surfaces/the inside of the packaging andensuring sterile conditions if desired. This may be embodied by weldingthe outer part of the opening during production of the packaging. Theend that is being used for the filling of the device is then cut shortlybefore the filling of the device.

In one embodiment, the opening may extend over a substantial part of onesurface of the packaging, for example by one of the walls beingtemporarily detached/not yet connected to the packaging. In this case,the opening is closed after filling the packaging by connecting thesecond wall to the first wall, for example by means of welding.

In one embodiment, the walls are quadrilateral and connected with eachother along at least three sides and that the opening for filling thepackaging is arranged in the fourth edge.

In one embodiment, an opening for draining air is arranged in the sameside edge of the periphery of the packaging as the opening for fillingthe packaging.

As the shape of the walls decides the shape of the packaging, the wallsare in one embodiment shaped in such a way that effective handling, e.g.such as for cryopreservation procedures or long time storage in liquidnitrogen, for example for transport is facilitated.

In one embodiment the length of the walls are at least about 15 timeslonger than the width, preferably about 12 times longer than the width.

The walls may for example be shaped as sheets. The walls should besemi-rigid, i.e. have certain stiffness in order to be able to maintainthe parallelism between the walls, while also having a certainflexibility in order to be able to allow the biologic material toexpand.

The walls may be connected to each other by means of welding, forexample pulse welding, gluing, folding, or a combination of thesesmethods.

The walls are connected to each other in at least a central connectionarea, for example by welding. The connected area may be a narrowsection, for example along the centre line/axis of the walls, or along anumber of lines. The connection area may also be constituted by a numberof point connections or a number of larger connection areas. Having morethan one connection area is particularly useful if the packaging isrelatively large. If the walls have an elongated shape, the centralconnection area may be along the longitudinal centre axis/line. Thecentral connection area is in most embodiments separate from theperiphery, allowing the biological material to flow between theperiphery and the central connection area. In the case of a number ofconnection areas, the connection areas may be separate, allowing thebiological material to flow also between and/or round the connectionareas.

The shape and size of the central connection area can vary according tothe shape and size of the walls and thus the packaging. A largerpackaging may need a higher number of connection areas and/or largerconnection areas. The connection of the walls in this central areaensures that the walls stay parallel after filling of the packaging andthrough subsequent processing of the packaging, thus ensuring the evenlyand sparsely distribution of the biological material. With parallel isin this description meant that the thickness over the packaging varieslittle and/or do not exceed a required/desired thickness. For examplemay the maximum thickness be 1 mm or 2 mm. The distance between theconnection areas must be adapted to the allowable thickness expansion ofthe packaging when filled. In one embodiment the distance between theconnection areas are less than 3 mm, or less than 2 mm.

The shape and arrangement of the central connection area may also beadapted for guiding the flow of biological material during filling ofthe packaging. For example may one or several longitudinal shapedcentral connection areas form channels inside the packaging. Suchchannels may lead the flow of biological material in a desired path, forexample to ensure complete filling of the packaging and drainage of airthrough an air draining opening.

In one embodiment the packaging comprises a funnel, for example arrangedin one side. The funnel may be arranged in the opening or replacing theopening, thus being the opening.

In one embodiment of the invention, the opening is arranged to be closedsubsequent to the filling, for example by pulse welding. There may alsoor alternatively be provided a closing device in the end, such as aplug.

In one embodiment, the external thickness of the packaging in emptystate is no larger than about 2.5 mm, preferably 1.3 mm.

The material used for the manufacturing of the packaging should be leakproof and impermeable.

The packaging is in one embodiment made of a biocompatible, non-toxicmaterial of food grade.

Examples of materials for making the packaging are: polyethylenterephtalate glycol (PETG), polyethylene, thermoplastic polyesther,(PET) polyvinyl chloride (PVC) or high securing ionomeric resine (IR),PEG, PE, or any combinations thereof.

In one embodiment, at least one of the walls is made of PET, and theinner surface of said walls are coated with PE.

In still another embodiment, one of the sheets/walls is made of PET andthe other opposite sheet/wall is made of PE or vice versa. The use ofboth PET and PE in the production of the device facilitates the closingof the device by pulse welding subsequent to filling of biologicalmaterial.

For identification purposes, the cryogenic device may furthermore beprovided with suitable identification means such as combinations ofcodes and colours, barcodes and/or alphanumeric codes. Theidentification marks or systems must furthermore be resistant to longterm storing in e.g. liquid nitrogen.

In one embodiment, a number of packagings are connected to each otheralong their sides and side by side, thus forming a packaging sheet. Thecharacteristics of each of the packagings are as described above.

The number of packagings in one packaging sheet can vary depending onthe needed volume or which type of biologic material that should bepackaged.

The connection of the packagings may be performed during production, forexample by provding two parallel films which will constitute the wallsof the packagings and forming the individual packagings by providingwelds or other connections between the two films in the areasconstituting the sides and the central connection area. There may alsobe provided perforations between the individual packagings in order tofacilitate separation of the packagings.

After being manufactured, the packagings of the invention may besterilized, for example by using gamma rays or by ionization or othersuitable methods well known to the person skilled in the art. Then thepackagings are packed in a manner to avoid contamination.

In one embodiment the packaging for cryopreservation of biologicalmaterial is filled by means of the following steps:

filling the biological material between two substantially parallel wallsconnected to each other along a substantially length of theircircumference through an opening,

closing the opening.

The filling of biologic material between the walls may be done byconventional methods well known to the person skilled in the art. Thefilling may be performed manually, for example by an operator using asyringe or cannula, or automatic. Furthermore, the filling of apackaging sheet may preferably be done in one operation. The filling ofall individual packagings may be performed simultaneously by using anautomatic filling apparatus. In one embodiment, the apparatus directseveral cannulas into the packagings and a predetermined volume ofsemen, e.g. 5 ml or 7 ml, is injected into each. Then the cannulas aredrawn out and the openings are closed by e.g. pulse welding. The sheetmay then be cut into separate packagings prior to further handling,storing etc.

In one embodiment, the biological material is filled between theparallel walls with a controlled flow rate.

The packagings according to the various embodiments of the invention mayalso be filled by applying suction to one end and then close the endthereafter in the same way as the filling of conventional cryogenicstraw. There may also be arranged suction in one opening of thepackaging while another opening is used for filling.

Subsequent to the filling of the biologic material, the packaging isclosed, e.g. by autogenic heat sealing at one or both ends using sealingequipment well known to the person skilled in the art, such as bymelting the end together by welding, for example ultrasound welding,high frequency welding, contact welding, etc. . Also, the packaging maybe provided with a closing device in the end, such as a plug used toclose the ends of common cryogenic straw. The end must then be adjustedto fit a suitable plug. Such adjustments are well within the knowledgeof the person skilled in the art.

The invention will now be described in more detail by means of anexample and by reference to the accompanying figures.

FIG. 1 shows one embodiment of the invention comprising a packagingsheet

FIG. 2 shows an example of an opening for filling a package according tothe invention.

FIG. 3 shows another embodiment of a packaging according to theinvention.

FIG. 1 shows one embodiment of the invention comprising a packagingsheet 10 comprising four packagings 11 for cryopreservation ofbiological material. Each of the packagings 11 comprise twosubstantially parallel walls 12, 13 connected to each other along asubstantially part of their periphery 14 and have an opening 15 forfilling the packaging. The walls are further connected to each other ina central area 16.

In this particular embodiment, the walls 12, 13, and thus the packagings11, are quadrilateral and elongated and the opening 15 is arranged inone of the end sides 17. The walls 12, 13 are connected along the threeremaining sides 18, 19, 20.

The central connection area 16 is in this embodiment an elongated areareaching over a substantial length along the longitudinal central axisof the packaging.

The packagings 11 are connected to each other along their longitudinalsides 18, 19 and thus arranged side by side. In this embodiment, thereare provided a perforation 21 between the individual packagings 11enabling separation of the packagings before or after filling.

FIG. 2 shows details of an embodiment of an opening 25 in a packagingaccording to the invention. The opening 25 is in this embodiment shapedas a funnel 26 in order to facilitate the filling of the packaging. FIG.2 b is a longitudinal cross section of the device and illustrates theprofile of the packaging along the opening 25, the parallel walls havinga distance between them at 27 and 28, and the central connection area 16where the walls are connected, thus having no distance between them.

FIG. 3 shows one embodiment of a packaging 30 according to theinvention. In this embodiment, the packaging 30 comprises twosubstantially parallel walls 31, 32, connected to each other along asubstantially part of their periphery 33. The packaging 30 has anopening 34 for filling the packaging and an opening 35 for draining air,the openings extending through the periphery of the packaging. Thepacking 30 has a rectangular form and the opening 34 for filling thepackaging and the opening 35 for draining air are located in one end ofthe packaging. The walls 31, 32 are connected in a central areaconstituted by two longitudinal connection areas 36, 37. Thelongitudinal connection areas 36, 37 are in this embodiment arranged inparallel with each other and with the long sides of the periphery 33 ofthe packaging, thus forming three separate “channels” 38, 39, 40 in thepackaging 30. The longitudinal connection areas are located offset fromthe location of the openings 34, 35 for filling the packaging and fordraining air, respectively. The longitudinal connection areas 36, 37 arenarrow, such that the area of the walls which are not connected arelarger than the total connection area. As an example, the breadth of thelongitudinal connection areas 36, 37 may be of the same magnitude as thebreadth of the connected periphery, for example 5 mm. The breadth of thetotal packaging may for example be 40-50 mm and the length may be300-320 mm.

The longitudinal connection areas 36, 37 have in this embodimentslightly different length, the longest connection area 37 being arrangedclosest to the opening 34 for filling the packaging and reaching nearertowards the packaging end with the two openings. With the connectionarea 37 being located offset to the right from the opening 34 forfilling the packaging, the biological material will flow into the firstchannel 38 and subsequently into the second 39 and third 40 channel fromthe other end of the packaging. In this way, any air in the packagingwill be pushed towards the opening 35 for draining air, thus ensuringthat little or no air is left in the packaging after filling.

In other embodiments, the longest connection area may reach to and beconnected to the connected periphery of the packaging or the twoconnection areas may have the same length.

Other embodiments are also possible, for example where the openings arearranged in the long edge of the packaging and the central connectionareas are a number of longitudinal areas parallel to the short edges.

1. Packaging for biological material characterised in that it comprisestwo substantially parallel walls connected to each other along asubstantially length of their periphery and at least one connection areain a central area of the packaging, the connection area providingcontact between the two parallel walls and being shaped to ensure thatthe walls stay parallel after filling.
 2. Packaging according to claim1, characterised in further comprising an opening for draining air. 3.Packaging according to claim 1, characterised in that the walls arequadrilateral and connected with each other along at least three sidesand that the opening is arranged in the fourth edge for filling thepackaging.
 4. Packaging according to claim 1, characterised in that thewalls are shaped as semi-rigid sheets.
 5. Packaging according to claim1, characterised in that it comprises a funnel, for example arranged inone side.
 6. Packaging according to claim 1, characterised in that itcomprises at least one opening for filling the packaging.
 7. Packagingaccording to claim 6, characterised in that the opening is a funnel. 8.Packaging according to claim 6, characterised in that the opening is ina closed state prior to filling.
 9. Packaging according to claim 6,characterised in that that the opening is arranged to be closedsubsequent to the filling, for example by pulse welding.
 10. Packagingaccording to claim 1, characterised in that the walls are separated by adistance between the inner surface of the two walls which is about 2 mm,more preferably about 1,1 mm, even more preferably about 1.0 mm, mostpreferably about 0,95 mm.
 11. Packaging according to claim 1,characterised in that the external thickness of the packaging is nolarger than about 2.4 mm, preferably 1.35 mm.
 12. Packaging according toclaim 1, characterised in that it is made of a biocompatible, non-toxicmaterial of food grade.
 13. Packaging according to claim 1,characterised in in that it is made of polyethylen terephtalate glycol(PETG), polyethylene, thermoplastic polyesther, (PET) polyvinyl chloride(PVC) or high securing ionomeric resine (IR), or any combinationsthereof.
 14. Packaging according to claim 1, characterised in it is madeof (PETG), PEG or PE.
 15. Packaging according to claim 1, characterisedin in that at least one of the walls is made of PET, and wherein theinner surface of said walls are coated with PE.
 16. Packaging accordingto claim 1, characterised in that a number of packagings are connectedto each other along their sides and side by side, thus forming apackaging sheet.
 17. Packaging according to claim 1, characterised inthat the two walls are connected in a number of points in a centralarea.
 18. Packaging according to claim 1, characterised in that the twowalls are connected in a number of areas distributed over the area ofthe walls.
 19. Method for filling a packaging for cryopreservation ofbiological material, characterised in that it comprises filling thebiological material between two substantially parallel walls connectedto each other along a substantially length of their circumferencethrough an opening while draining air through a second opening, andclosing the opening.
 20. Method according to claim 19, characterised inthat the closing of the opening is done by pulse welding.
 21. Methodaccording to claim 19, characterised in that the filling is performed bymeans of a cannula.
 22. Method according to claim 19, characterised inthat the opening is constituted by the absence of one wall.
 23. Methodaccording to claim 19, characterised in that the closing of the openingis done by placing a wall over the opening and connecting the walls. 24.(canceled)